Methods for treating nlrp3 inflammasome-associated diseases, and methods of identifying agents useful therefor

ABSTRACT

Provided herein are methods of treating NLRP3 inflammasome-associated diseases and disorders. Also, disclosed are methods for screening for agents useful in such methods.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. Ser. No. 16/451,674, filed onJun. 25, 2019, which claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Ser. No. 62/690,175, filed on Jun. 26, 2018, the entirecontents of each of which are incorporated herein by reference.

GRANT INFORMATION

This invention was made with government support under Grant Nos.AI043477 and ES010337 awarded by the National Institutes of Health. TheUnited States government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 12, 2020, isnamed 114198-4082_SL.txt and is 8 kilobytes in size.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates generally to inflammation and more specifically tomethods and compositions for preventing NLRP3 inflammasome activation totreat inflammation and degenerative diseases.

Background Information

Chronic inflammation is involved in many different diseases includingAlzheimer's disease, Parkinson's disease, Crohn's disease, myositis,inflammatory bowel disease, osteoarthritis, gout, rheumatoid arthritis,ankylosing spondylitis, uveitis, myositis, diverticulitis, dermatitis,colitis, autoimmune diseases, atherosclerosis, asthma, and cancer (Grosset al., 2011; Grivennikov, Greten and Karin, 2010). Inflammation isinitiated upon sensing of pathogen (PAMP)- or damage (DAMP)-associatedmolecular patterns via pattern recognition receptors (PRR) (Gross etal., 2011; Kotas and Medzhitov, 2015). Amongst PRR, Nod-like receptorpyrin domain containing 3 (NLRP3) is unique in its ability to undergoactivation in response to highly diverse extracellular stimuli, many ofwhich, such as ATP and uric acid, are associated with tissue damage andcan trigger sterile inflammation, which stimulates damage repair (Karinand Clevers, 2016; Zhong et al., 2016). Of note, NLRP3 is the sensorprotein of an inflammasome complex that also contains the scaffoldprotein ASC and caspase-1, collectively referred to as the NLRP3inflammasome (Gross et al., 2011).

Although the subject of intense efforts in academia and pharma alike, noeffective strategies for inhibition of NLRP3 inflammasome activation andIL-1β production are clinically available. Moreover, the mechanism thattriggers NLRP3 inflammasome activation remains poorly understood. A needtherefore exists for treatments that prevent or ameliorate NLRP3inflammasome-dependent diseases and attenuate their progression.

SUMMARY OF THE INVENTION

The present invention is based on the observation that activation of theNLRP3 inflammasome depends on a specific signal generated by oxidizedmitochondrial (mt) DNA and interference with the generation of thissignal is capable of inhibiting NLRP3 inflammasome-driven inflammation.Accordingly, in one aspect, the invention provides a method of treatingNLRP3 inflammasome-associated inflammatory and/or degenerative diseasesor cancers in a subject. The method includes administering to a subjectin need thereof an effective amount of an inhibitor of NLRP3inflammasome activation. In various embodiments, the inhibitor of NLRP3inflammasome activation inhibits interferon regulatory factor 1 (IRF 1)activity or expression and/or cytidine monophosphate kinase 2 (CMPK2)activity or expression. In various embodiments, the subject is a mammal,such as a human. In various embodiments, the inhibitor of CMPK2 activityor expression is a small molecule, peptide, antisense oligonucleotide,RNA hairpin, guide DNA, antibody or an antibody fragment. In variousembodiments, the inhibitor of CMPK2 activity or expression is aninhibitory nucleic acid that inhibits the expression of CMPK2. Invarious embodiments, the inhibitory nucleic acid is selected from thegroup consisting of siRNA, shRNA, gRNA, oligonucleotides, antisense RNAor ribozymes that inhibit CMPK2 synthesis. In various embodiments, theinhibitory nucleic acid is administered via a viral vector, a liposomeor a nanoparticle. In various embodiments, the NLRP3inflammasome-associated inflammatory and/or degenerative disease isselected from the group consisting of cancer (especially lung cancer),lupus, gout, rheumatoid arthritis, osteoarthritis, ankylosingspondylitis, uveitis, Alzheimer's disease, Parkinson's disease,cryopyrin-associated periodic syndromes, nonalcoholic steatohepatitis(NASH), type 2 diabetes, atherosclerosis, macular degeneration, andgeographical retinopathy.

In another aspect, the invention provides a method of inhibiting NLRP3inflammasome activation in a subject. The method includes administeringto the subject an effective amount of an inhibitor of CMPK2 activity orexpression. In various embodiments, the inhibitor of CMPK2 activity orexpression is a small molecule, peptide, antisense oligonucleotide,antibody or an antibody fragment. In various embodiments, the inhibitorof CMPK2 activity or expression is an inhibitory nucleic acid thatinhibits the expression of CMPK2 or inhibits CMPK2 synthesis. In variousembodiments, the inhibitor nucleic acid is selected from the groupconsisting of siRNA, shRNA, gRNA, oligonucleotides, antisense RNA orribozymes that inhibit CMPK2 synthesis. In various embodiments, theinhibitory nucleic acid is administered via a viral vector, liposome ora nanoparticle.

In another aspect, the invention provides a method of identifying anagent that inhibits NLRP3 inflammasome activation through the targetingof either IRF1 or CMPK2. The method includes contacting a sample ofcells with at least one test agent, wherein a decrease in CMPK2 activityor expression in the presence of the test agent as compared to CMPK2activity or expression in the absence of the test agent identifies theagent as useful for inhibiting NLRP3 inflammasome activation by virtueof its ability to reduce CMPK2 activity or expression. In variousembodiments, the test agent is a small molecule, peptide, antisenseoligonucleotide, antibody or an antibody fragment. In variousembodiments, the method may be performed in a high throughput format,such as contacting samples of cells of a plurality of samples with atleast one test agent. In various embodiments, the plurality of samplesmay be obtained from a single subject or from different subjects. Inother embodiments, the CMPK2 targeting agent is selected from a libraryof known nucleotide kinase inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F are graphical and pictorial diagrams showing thatmitochondrial (mt) DNA, whose replication is stimulated by occupancy ofToll-like receptors (TLR), is needed for activation of the NLRP3inflammasome. FIG. 1A shows that shCtrl or shNlrp3 transducedTfam^(Δmye) (i.e., TFAM-deficient) bone marrow derived macrophages(BMDM) were incubated with LPS, synthetic Ox-mtDNA or LPS+syntheticOx-mtDNA. The release of IL-1β to culture supernatants was measured 4hrs later. Data are averages±s.d. (n=3). FIG. 1B shows that relativeamounts of total mtDNA were quantified by qPCR using primers specificfor mtDNA (cytochrome c oxidase I, and D-loop) and nuclear (n)DNA (18Sand Tert) in WT BMDM before and after LPS (200 ng/ml) treatment. Dataare averages±s.d. (n=3). FIG. 1C shows representative immunofluorescent(IF) staining of EdU-labeled newly synthesized mtDNA in WT BMDM beforeand after LPS stimulation. Scale bars 5 μm. Images representative of 3independent experiments. FIG. 1D shows relative levels of total mtDNA inimmortalized BMDM (iBMDM) transduced with shCtrl or shPolg RNAs beforeand after LPS treatment. Data are averages±s.d. (n=3). FIG. 1E showsrelative amounts of total mtDNA in WT, Myd88^(−/−) and Trif^(−/−) beforeand after LPS stimulation were qPCR determined. Data are averages±s.d.(n=3). FIG. 1F shows relative amounts of total mtDNA in WT andIfnar1^(−/−) BMDM before and after LPS stimulation were qPCR determined.Data are averages±s.d. (n=3).

FIGS. 2A-2G are pictorial and graphical diagrams showing that IRF1controls mtDNA synthesis, Ox-mtDNA generation and NLRP3 inflammasomeactivation. FIG. 2A shows relative amounts of total mtDNA that werequantified by qPCR using primers specific for mtDNA (D-loop) and nDNA(Tert) in WT and Irf1^(−/−) BMDM that were stimulated with LPS asindicated. The amounts of mtDNA in these cells were quantified by qPCRusing the medicated primers. Data are averages±s.d. (n=3). FIG. 2B showsrelative amounts of total mtDNA that were quantified by qPCR usingprimers specific for mtDNA (cytochrome c oxidase I) and nDNA (18S) in WTand Irf1^(−/−) BMDM were stimulated with LPS as indicated. The amountsof mtDNA in these cells were quantified by qPCR using the indicatedprimers. Data are averages±s.d. (n=3). FIG. 2C shows representativeimages showing EdU incorporation into mtDNA in WT and Irf1^(−/−) BMDMincubated without or with LPS. Scale bars 5 FIG. 2D shows quantificationof the fraction of cells showing mtDNA replication as determined in FIG.2C. Averages±s.d. (n=257˜313). FIG. 2E shows immunoblot (TB) analysis ofcleaved caspase-1 (Casp1 p20) released into culture supernatants of WTand Irf1^(−/−) BMDM that were treated with LPS plus indicatedinflammasome activators. W=WT; K=Irf1^(−/−). FIG. 2F shows IL-1β inculture supernatants of LPS-primed WT and Irf1^(−/−) BMDM that weretreated with LPS and the indicated NLRP3 activators. Data areaverages±s.d. (n=3). FIG. 2G shows amounts of 8-OH-dG in mtDNA isolatedfrom LPS-primed WT and Irf1^(−/−) BMDM that were treated with LPS andthe indicated NLRP3 activators. Data are averages±s.d. (n=3).

FIGS. 3A-3F are pictorial and graphical diagrams showing that CMPK2controls mtDNA synthesis, Ox-mtDNA generation and NLRP3 inflammasomeactivation. FIG. 3A shows relative amounts of Cmpk2 mRNA in WT andIrf1^(−/−) BMDM before and after LPS stimulation. Data are averages±s.d.(n=3). FIG. 3B shows immunoblot (IB) analysis of CMPK2 in WT andIrf1^(−/−) BMDM before and after LPS stimulation. The arrow points tothe CMPK2 band. FIG. 3C shows IB analysis of Casp1 p20 and mature IL-1β(p17) in supernatants of shCtrl (W) and shCmpk2 (K) BMDM that werestimulated with LPS plus the indicated inflammasome agonists. FIG. 3Dshows IL-1β secretion by LPS-primed shCtrl and shCmpk2 BMDM that weretreated with the indicated NLRP3 agonists. Data are averages±s.d. (n=3).FIG. 3E shows relative amounts of total mtDNA in shCtrl and shCmpk2 BMDMbefore and after LPS stimulation. Data are averages±s.d. (n=3). FIG. 3Fshows amounts of 8-OH-dG (oxidized deoxy-guanosine) in mtDNA isolatedfrom LPS-primed WT and Irf1^(−/−) BMDM that were treated with indicatedNLRP3 activators. Data are averages±s.d. (n=3).

FIGS. 4A-4D are graphical diagrams showing CMPK2 catalytic activity isrequired for IRF1-dependent NLRP3 inflammasome activation. FIG. 4A showsrelative amounts of total mtDNA in LPS-treated WT and Irf1^(−/−) BMDMthat were transduced with WT Cmpk2 or empty lentiviruses. Data areaverages±s.d. (n=3). FIG. 4B shows IL-1β secretion by CMPK2lentivirus-transduced WT and Irf1^(−/−) BMDM that were stimulated withLPS+ATP or LPS+MSU. Data are averages±s.d. (n=3). FIG. 4C shows relativeamounts of total mtDNA in LPS-treated WT and Irf1^(−/−) BMDM that weretransduced with either WT Cmpk2 or Cmpk2(D330A) lentiviruses. Data areaverages±s.d. (n=3). FIG. 4D shows IL-1β secretion by WT Cmpk2 orCmpk2(D330A) -reconstituted WT and Irf1^(−/−) BMDM that were stimulatedas indicated. Data are averages±s.d. (n=3).

FIG. 5 is a pictorial diagram showing that newly synthesized mtDNAassociated with the NLRP3 inflammasome upon mitochondrial damage. Asshown, representative IF images of EdU-prelabeled BMDM were co-stainedwith ASC and DAPI before and after stimulation with LPS plus theindicated inflammasome activators. Scale bars, 5 μm.

FIGS. 6A-6H are pictorial and graphical diagrams showing that TFAM isrequired for maintenance of mtDNA, generation of Ox-mtDNA, andactivation of the NLRP3 inflammasome. FIG. 6A shows that relativeamounts of total mtDNA in Tfam^(F/F) and Tfam^(Δmye) BMDM werequantified by qPCR using primers specific for mtDNA (cytochrome coxidase I) and nDNA (18S). Data are averages±s.d. (n=3). FIG. 6B showsthat relative amounts of total mtDNA in Tfam^(F/F) and Tfam^(Δmye) BMDMwere quantified by qPCR using primers specific for mtDNA (D-loop) andnDNA (Tert). Data are averages±s.d. (n=3). FIG. 6C shows amounts of8-OH-dG in mtDNA isolated from Tfam^(F/F) and Tfam^(ΔMye) BMDM that werestimulated with LPS plus various NLRP3 activators. Data areaverages±s.d. (n=3). FIG. 6D shows D3 analysis of Casp1 p20 and matureIL-1β (p17) in culture supernatants of Tfam^(F/F) (W) and Tfam^(ΔMye)(K) BMDM that were stimulated with LPS plus various inflammasomeactivators. Results are typical of three separate experiments. FIG. 6Eshows IB analysis of pro-IL-1β, NLRP3, ASC, and pro-Casp1 in lysates ofTfam^(F/F) and Tfam^(ΔMye) BMDM before and after LPS priming. Resultsare typical of three separate experiments. FIG. 6F shows amounts ofIL-1β in culture supernatants of LPS-primed Tfam^(F/F) and Tfam^(ΔMye)BMDM that were stimulated with various NLRP3 activators. Data areaverages±s.d. (n=3). FIG. 6G shows amounts of TNF in culturesupernatants of LPS-primed Tfam^(F/F) and Tfam^(ΔMye) BMDM that werestimulated with various NLRP3 agonists. Data are averages±s.d. (n=3).FIG. 6H shows representative IF images of WT BMDM that were co-stainedwith 8-OH-dG, ASC and DAPI before and after stimulation with LPS plusthe indicated inflammasome agonists. Scale bars, 5 μm.

FIGS. 7A-7G are pictorial and graphical diagrams showing that IRF1 doesnot affect inflammasome subunit expression nor NLRP3 agonist-inducedmitochondrial damage. FIG. 7A shows relative amounts of Irf1 mRNA in WT,Trif^(−/−) and MyD88^(−/−) BMDM before and after LPS stimulation. Dataare averages±s.d. (n=3). FIG. 7B shows IB analysis of IRF1 in lysates ofWT, Trif^(−/−) and MyD88^(−/−) BMDM before and after LPS priming.Results are typical of 3 separate experiments. FIG. 7C shows relativeamounts of Irf1 mRNA in WT and Ifnar1^(−/−) BMDM before and after LPSpriming. Data are averages±s.d. (n=3). FIG. 7D shows IB analysis ofpro-IL-1β, NLRP3, ASC, and pro-Casp1 in lysates of WT and Irf1^(−/−)BMDM before and after LPS priming. Results are typical of 3 separateexperiments. FIG. 7E shows TNF secretion by LPS-primed WT and Irf1^(−/−)BMDM that were stimulated with various NLRP3 activators. Data areaverages±s.d. (n=3). FIG. 7F shows NLRP3 activator-induced changes inmitochondrial membrane potential (ψm) in LPS-primed WT and Irf1^(−/−)BMDM were measured by TMRM fluorescence. Data are averages±s.d. (n=3).FIG. 7G shows relative amounts of mtROS were measured by MitoSOXfluorescence in LPS-primed WT and Irf1^(−/−) BMDM after stimulation withvarious NLRP3 activators. Data are averages±s.d. (n=3).

FIGS. 8A-8D are pictorial and graphical diagrams showing thatLPS-induced CMPK2 expression depends on the signaling proteins TRIF andIFNAR. FIG. 8A shows relative amounts of Cmpk2 mRNA in WT and Trif^(−/−)BMDM before and after LPS stimulation. Data are averages±s.d. (n=3).FIG. 8B shows relative amounts of Cmpk2 mRNA in WT and Ifnar1^(−/−) BMDMbefore and after LPS stimulation. Data are averages±s.d. (n=3). D3analysis of CMPK2 in lysates of WT and Ifnar1^(−/−) BMDM before andafter LPS stimulation. Results are typical of three separateexperiments. FIG. 8D shows the Cmpk2 promoter contains IRF1 bindingsites (site 1, SEQ ID NO: 3; site 2, SEQ ID NO: 4; site 3, SEQ ID NO:5).

FIG. 9 is a series of graphical diagrams showing that expression ofmitochondrial dNTP salvage pathway enzymes and DNA polymerase γ. FIG. 9shows relative mRNA amounts of dGK, Tk2, Ak2, Nme4, and Polg in WT BMDMbefore and after LPS stimulation. Data are averages±s.d. (n=3).

FIGS. 10A-10C are pictorial and graphical diagrams showing that CMPK2does not affect inflammasome subunit expression and NLRP3agonist-induced mitochondrial damage. FIG. 10A shows IB analysis ofCMPK2, pro-IL-1β, NLRP3, ASC, and pro-Casp1 in lysates of WT (shCtrl)and CMPK2-deficient (shCmpk2) BMDM before and after LPS priming. Resultsare typical of 3 separate experiments. FIG. 10B shows NLRP3activator-induced changes in ψm in LPS-primed shCtrl and shCmpk2 BMDMwere measured by TMRM fluorescence. Data are averages±s.d. (n=3). FIG.10C shows relative amounts of mtROS measured by MitoSOX fluorescence inLPS-primed shCtrl and shCmpk2 BMDM after stimulation with various NLRP3activators. Data are averages±s.d. (n=3).

FIGS. 11A-11D are graphical diagrams showing that NME4 is needed forNLRP3 inflammasome activation. FIG. 11A shows relative amounts of totalmtDNA in shCtrl and shNme4 BMDM before and after LPS priming. Data areaverages±s.d. (n=3). FIG. 11B shows amounts of 8-OH-dG in mtDNA isolatedfrom shCtrl and shNme4 BMDM that were stimulated or not with LPS plusvarious NLRP3 activators. Data are averages±s.d. (n=3). FIG. 11C showsamounts of IL-1β in supernatants of LPS-primed shCtrl and shNme4 BMDMthat were stimulated with various inflammasome activators. Data areaverages±s.d. (n=3). FIG. 11D shows amounts of TNF in supernatants ofLPS-primed shCtrl and shNme4 BMDM that were stimulated with variousinflammasome activators. Data are averages±s.d. (n=3).

FIGS. 12A and 12B are graphical diagrams showing that expression ofCMPK2 restores NLRP3 inflammasome activation in IRF1-deficientmacrophages. IB analysis of CMPK2, pro-IL-1β, NLRP3, ASC, and pro-Casp1in lysates of WT and Irf1^(−/−) BMDM before and after transduction withthe WT Cmpk2 lentivirus. FIG. 12A shows NLRP3 activator-induced changesin ψm in LPS-primed CMPK2-transduced WT and Irf1^(−/−) BMDM weremeasured by TMRM fluorescence. Data are averages±s.d. (n=3). FIG. 12Bshows relative amounts of mtROS measured by MitoSOX fluorescence inLPS-primed CMPK2-transduced WT and Irf1^(−/−) BMDM before and afterstimulation with ATP or nigericin. Data are averages±s.d. (n=3). IBanalysis of Casp1 p20 and mature IL-1β (p17) in supernatants ofLPS-primed CMPK2-transduced WT and Irf1^(−/−) BMDM before and afterstimulation with various NLRP3 activators.

FIGS. 13A-13F are graphical diagrams showing that IRF1 controls mtDNAreplication and NLRP3 inflammasome activation in vivo. FIG. 13A shows12-week old WT or Irf1^(−/−) mice were i.p. injected with LPS and theirsera collected 3 hrs later and analyzed by ELISA for IL-1β. Results areaverages±s.d. (n=8). FIG. 13B shows 12-week old WT or Irf1^(−/−) micewere i.p. injected with LPS and their sera collected 3 hrs later andanalyzed by ELISA for TNF. Results are averages±s.d. (n=8). FIG. 13Cshows survival of WT or Irf1^(−/−) mice that were i.p. injected with LPS(50 mg/kg), n=10-11. FIG. 13D shows relative amounts of total mtDNA inperitoneal infiltrates of WT or Irf1^(−/−) mice and before and after LPS(50 mg/kg) injection. Data are averages±s.d. (n=3). FIG. 13E showsperitoneal IL-1β in WT or Irf1^(−/−) mice 4 hrs after i.p. injection ofalum (300 μg) or PBS. n=3-6. FIG. 13F shows proteinuria was measured inurine of WT or Irf1^(−/−) female mice before and 12 hrs after i.p.injection with 300 mg/Kg folic acid. Data are shown as mean±s.e.m. of5-10 mice. **, p<0.01. Results are representative of kidney extractsfrom three mice per group. Alum-induced peritoneal infiltration ofneutrophils (CD11b⁺Ly6G⁺F4/80⁻). Biochemical analysis of protein,creatine, BUN and electrolites levels in sera of the mice. Data aremean±s.e.m. of 5-10 mice. Representative H&E staining of kidney sectionsfrom treated mice. IB analysis of casp1 p20, mature IL-1β, IRF1, CMPK2and tubulin in the whole kidney extracts from mice treated as in h.Results are representative of kidney extracts from three mice per group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the observation that persistentactivation of the NLRP3 inflammasome results in uncontrolledinflammation that contributes to the pathogenesis of many chronic andacute inflammatory and degenerative diseases. Thus, without being boundby theory, NLRP3 appears to be the key sensor of tissue damage andmediator of sterile inflammation, playing a central role in thepathogenesis of acute and chronic inflammatory diseases.

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

The term “comprising,” which is used interchangeably with “including,”“containing,” or “characterized by,” is inclusive or open-ended languageand does not exclude additional, unrecited elements or method steps. Thephrase “consisting of” excludes any element, step, or ingredient notspecified in the claim. The phrase “consisting essentially of” limitsthe scope of a claim to the specified materials or steps and those thatdo not materially affect the basic and novel characteristics of theclaimed invention. The present disclosure contemplates embodiments ofthe invention compositions and methods corresponding to the scope ofeach of these phrases. Thus, a composition or method comprising recitedelements or steps contemplates particular embodiments in which thecomposition or method consists essentially of or consists of thoseelements or steps.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

The term “subject” as used herein refers to any individual or patient towhich the subject methods are performed. Generally the subject is human,although as will be appreciated by those in the art, the subject may bean animal. Thus other animals, including mammals such as rodents(including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits,farm animals including cows, horses, goats, sheep, pigs, etc., andprimates (including monkeys, chimpanzees, orangutans and gorillas) areincluded within the definition of subject.

As used herein, a “non-human mammal” may be any animal as long as it isother than human, and includes a transgenic animal and animals for whicha production method of ES cells and/or iPS cells has been established.For example, rodents such as mouse, rat, hamster, guinea pig, rabbit,swine, bovine, goat, horse, sheep, dog, cat, or monkey are envisioned asnon-human mammals.

The terms “administration” or “administering” are defined to include anact of providing a compound or pharmaceutical composition of theinvention to a subject in need of treatment. The phrases “parenteraladministration” and “administered parenterally” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinaland intrasternal injection and infusion. The phrases “systemicadministration,” “administered systemically,” “peripheraladministration” and “administered peripherally” as used herein mean theadministration of a compound, drug or other material other than directlyinto the central nervous system, such that it enters the subject'ssystem and, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration or administration via intranasaldelivery.

As used herein, an “effective amount” is an amount of a substance ormolecule sufficient to effect beneficial or desired clinical resultsincluding alleviation or reduction in any one or more of the symptomsassociated with NLRP3 inflammasome-driven inflammation such as, but notlimited to, cancer, lupus, gout, rheumatoid arthritis, osteoarthritis,ankylosing spondylitis, uveitis, Alzheimer's disease, Parkinson'sdisease, cryopyrin-associated periodic syndromes, nonalcoholicsteatohepatitis (NASH), type 2 diabetes, atherosclerosis, maculardegeneration, and many more inflammatory and degenerative diseases. Forpurposes of this invention, an effective amount of a compound ormolecule of the invention is an amount sufficient to reduce the signsand symptoms associated with such disorders. In some embodiments, the“effective amount” may be administered before, during, and/or after anytreatment regimens for the above-mentioned diseases.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, treatment ofNLRP3 inflammasome-associated diseases or disorders.

As used herein, the term “genetic modification” is used to refer to anymanipulation of an organism's genetic material in a way that does notoccur under natural conditions. Methods of performing such manipulationsare known to those of ordinary skill in the art and include, but are notlimited to, techniques that make use of vectors for transforming cellswith a nucleic acid sequence of interest. Included in the definition arevarious forms of gene editing in which DNA is inserted, deleted orreplaced in the genome of a living organism using engineered nucleases,or “molecular scissors.” These nucleases create site-specificdouble-strand breaks (DSBs) at desired locations in the genome. Theinduced double-strand breaks are repaired through nonhomologousend-joining (NHEJ) or homologous recombination (HR), resulting intargeted mutations (i.e., edits). There are several families ofengineered nucleases used in gene editing, for example, but not limitedto, meganucleases, zinc finger nucleases (ZFNs), transcriptionactivator-like effector-based nucleases (TALEN), and the CRISPR-Cassystem.

As used herein, the term “test agent” or “candidate agent” refers to anagent that is to be screened in one or more of the assays describedherein. The agent can be virtually any chemical compound. It can existas a single isolated compound or can be a member of a chemical (e.g.,combinatorial) library. In one embodiment, the test agent is a smallorganic molecule. The term small organic molecule refers to anymolecules of a size comparable to those organic molecules generally usedin pharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). In certain embodiments, small organicmolecules range in size up to about 5000 Da, up to 2000 Da, or up toabout 1000 Da.

As used herein, the terms “sample” and “biological sample” refer to anysample suitable for the methods provided by the present invention. Inone embodiment, the biological sample of the present invention is atissue sample, e.g., a biopsy specimen such as samples from needlebiopsy (i.e., biopsy sample). In other embodiments, the biologicalsample of the present invention is a sample of bodily fluid, e.g.,serum, plasma, sputum, lung aspirate, urine, and ejaculate.

The term “antibody” is meant to include intact molecules of polyclonalor monoclonal antibodies, chimeric, single chain, and humanizedantibodies, as well as fragments thereof, such as Fab and F(ab′)2, Fvand SCA fragments which are capable of binding an epitopic determinant.Monoclonal antibodies are made from antigen containing fragments of theprotein by methods well known to those skilled in the art (Kohler, etal., Nature, 256:495, 1975). An Fab fragment consists of a monovalentantigen binding fragment of an antibody molecule, and can be produced bydigestion of a whole antibody molecule with the enzyme papain, to yielda fragment consisting of an intact light chain and a portion of a heavychain. An Fab′ fragment of an antibody molecule can be obtained bytreating a whole antibody molecule with pepsin, followed by reduction,to yield a molecule consisting of an intact light chain and a portion ofa heavy chain. Two Fab′ fragments are obtained per antibody moleculetreated in this manner. An (Fab′)2 fragment of an antibody can beobtained by treating a whole antibody molecule with the enzyme pepsin,without subsequent reduction. A (Fab′)2 fragment is a dimer of two Fab′fragments, held together by two disulfide bonds. An Fv fragment isdefined as a genetically engineered fragment containing the variableregion of a light chain and the variable region of a heavy chainexpressed as two chains. A single chain antibody (“SCA”) is agenetically engineered single chain molecule containing the variableregion of a light chain and the variable region of a heavy chain, linkedby a suitable, flexible polypeptide linker.

Reference herein to “normal cells” or “corresponding normal cells” meanscells that are from the same organ and of the same type as any of theabove-mentioned disease cell type. In one aspect, the correspondingnormal cells comprise a sample of cells obtained from a healthyindividual. Such corresponding normal cells can, but need not be, froman individual that is age-matched and/or of the same sex as theindividual providing the above-mentioned disease cells being examined.In another aspect, the corresponding normal cells comprise a sample ofcells obtained from an otherwise healthy portion of tissue of a subjecthaving an NLRP3-inflammasome inflammatory and/or degenerative disease.

The molecular mechanisms that control NLRP3 inflammasome are complex andinclude aberrant elements (Gross et al., 2011). Without being bound bytheory, upon ligand (e.g., oxidized mtDNA) binding NLRP3 is thought tounfold and expose its pyrin domain, which binds to the adaptor proteinASC (apoptosis associated spike-like protein) that recruits the effectormolecule pro-caspase-1 via CARD:CARD interactions to form a largecytosolic complex/aggregate—the NLRP3 inflammasome (Gross et al., 2011;Kotas and Medzhitov, 2015; Lu et al., 2014). Inflammasome assemblyresults in self cleavage and activation of caspase-1, which convertsimmature proinflammatory cytokines (e.g., pro-IL-1β and pro-IL-18) totheir mature forms (Gross et al., 2011). Although DAMP-induced NLRP3inflammasome activation is important for stimulation of tissue repairand regenerative processes (Karin and Clevers, 2016), aberrant NLRP3activation appears to be the key to many chronic diseases, includingcryopyrin-associated periodic syndromes, gout, Alzheimer's disease, type2 diabetes, atherosclerosis, lupus, macular degeneration and cancer(Heneka et al., 2014; Lamkanfi and Dixit, 2012; Martinon et al., 2009).Heretofore, the ligand that binds to NLRP3 to induce inflammasomeactivation and IL-1β production has not been identified.

NLRP3 inflammasome activation and production of bioactive IL-1β requiretwo functionally distinct steps: “priming” and “activation” (Gross etal., 2011; Zhong et al., 2016; Martinon et al., 2009). “Priming” entailsdirect engagement of Toll-like receptors (TLR) by PAMP and DAMP, such asLPS, resulting in rapid NF-KB activation, which leads to de novosynthesis of pro-IL-1β and upregulation of NLRP3 expression. Thesubsequent “activation” step is more mysterious, leading to NLRP3inflammasome assembly and eventual caspase-1 self-cleavage andactivation (Gross et al., 2011; Lu et al., 2014; Schroder and Tschopp,2010). A major difficulty in understanding “activation” is the abilityof numerous chemically and structurally diverse extracellular stimuli,often referred to as “NLRP3 agonists” or “NLRP3 activators”, whichinclude microparticles, microfibers, pore-forming toxins, ATP, and somepathogens, to trigger inflammasome assembly, although none of themdirectly binds NLRP3 (Gross et al., 2011; Latz et al., 2013). Onesolution to this conundrum is the proposal that all NLRP3 activatorsoperate through a common intracellular intermediate, most likely themitochondrion (Nakahira et al., 2011; Zhong et al., 2016; Zhou et al.,2011). Engaging different mechanisms, some of which involve plasmamembrane damage, K⁺ efflux and elevated intracellular Ca²⁺, NLRP3activators elicit a particular form of mitochondrial damage that causesrelease of fragmented mitochondrial (mt) DNA and increased production ofreactive oxygen species (ROS) that convert mtDNA to an oxidized form,Ox-mtDNA, which was suggested to serve as a direct NLRP3 ligand (Shimadaet al., 2012). The type of mitochondrial damage induced by NLRP3agonists is distinct from that induced by pro-apoptotic members of theBCL2 family, which enable cytochrome c release and activation of theAPAF complex that culminate in caspase-3, rather than caspase-1,activation (Jiang and Wang, 2004).

In contrast to the other known inflammasomes, activation of the NLRP3inflammasome can be achieved by a wide range of structurally dissimilarstimuli, including pathogens, pore-forming toxins, environmentalirritants, and endogenous DAMPs (Gross et al., 2011). Numerous moleculesmay trigger the formation and activation of the NLRP3 inflammasome.

By blocking the increase in NLRP3 inflammasome activation andproduction, the present disclosure demonstrates the ability to preventsuch diseases associated with aberrant or persistent NLRP3 inflammasomeactivation and production. Accordingly, in one aspect, the inventionprovides a method of treating NLRP3 inflammasome-associated inflammatoryand/or degenerative diseases in a subject in need thereof. The methodincludes administering to the subject an effective amount of aninhibitor of NLRP3 inflammasome activation. In various embodiments, theinhibitor of NLRP3 inflammasome activation may be an inhibitor ofinterferon regulatory factor 1 (IRF1) activity or expression and/orcytidine monophosphate kinase 2 (CMPK2) activity or expression. Invarious embodiments, the inhibitor of CMPK2 activity or expression issmall molecule, peptide, antisense oligonucleotide, guide RNA, shRNA,antibody or an antibody fragment.

In various embodiments, the inhibitor of CMPK2 activity or expression isan inhibitory nucleic acid that specifically inhibits expression ofCMPK2 and/or inhibits CMPK2 activation. As used herein, an “inhibitorynucleic acid” means an RNA, DNA, or a combination thereof thatinterferes or interrupts the translation of mRNA. Inhibitory nucleicacids can be single or double stranded. The nucleotides of theinhibitory nucleic acid can be chemically modified, natural orartificial. The terms “short-inhibitory RNA” and “siRNA” interchangeablyrefer to short double-stranded RNA oligonucleotides that mediate RNAinterference (also referred to as “RNA-mediated interference” or“RNAi”). The terms “small hairpin RNA” and “shRNA” interchangeably referto an artificial RNA molecule with a tight hairpin turn that can be usedto silence target gene expression via RNAi. RNAi is a highly conservedgene silencing event functioning through targeted destruction ofindividual mRNA by a homologous double-stranded small interfering RNA(siRNA) (Fire, A. et al., Nature 391:806-811 (1998)). Mechanisms forRNAi are reviewed, for example, in Bayne and Allshire, Trends inGenetics (2005) 21:370-73; Morris, Cell Mol Life Sci (2005) 62:3057-66;Filipowicz, et al., Current Opinion in Structural Biology (2005)15:331-41.

Methods for the design of siRNA or shRNA target sequences have beendescribed in the art. Among the factors to be considered include: siRNAtarget sequences should be specific to the gene of interest and haveabout 20-50% GC content (Henshel et al., Nucl. Acids Res., 32: 113-20(2004); G/C at the 5′ end of the sense strand; A/U at the 5′ end of theantisense strand; at least 5 A/U residues in the first 7 bases of the 5′terminal of the antisense strand; and no runs of more than 9 G/Cresidues (Ui-Tei et al., Nucl. Acids Res., 3: 936-48 (2004)).Additionally, primer design rules specific to the RNA polymerase willapply. For example, for RNA polymerase III, the polymerase thattranscribes from the U6 promoter, the preferred target sequence is5′-GN18-3′. Runs of 4 or more Ts (or As on the other strand) will serveas terminator sequences for RNA polymerase III and should be avoided. Inaddition, regions with a run of any single base should be avoided(Czauderna et al., Nucl. Acids Res., 31: 2705-16 (2003)). It has alsobeen generally recommended that the mRNA target site be at least 50-200bases downstream of the start codon (Sui et al., Proc. Natl. Acad. Sci.USA, 99: 5515-20 (2002); Elbashir et al., Methods, 26: 199-213 (2002);Duxbury and Whang, J. Surg. Res., 117: 339-44 (2004) to avoid regions inwhich regulatory proteins might bind. Additionally, a number of computerprograms are available to aid in the design of suitable siRNA and shRNAsfor use in suppressing expression of casp2 and/or inhibiting casp2synthesis.

Ribozymes that cleave mRNA at site-specific recognition sequences can beused to destroy target mRNAs, particularly through the use of hammerheadribozymes. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. Preferably, the target mRNA has the following sequence of twobases: 5′-UG-3′. The construction and production of hammerhead ribozymesis well known in the art.

Gene targeting ribozymes may contain a hybridizing region complementaryto two regions, each of at least 5 and preferably each 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides inlength of a target mRNA. In addition, ribozymes possess highly specificendoribonuclease activity, which autocatalytically cleaves the targetsense mRNA.

With regard to antisense, siRNA or ribozyme oligonucleotides,phosphorothioate oligonucleotides can be used. Modifications of thephosphodiester linkage as well as of the heterocycle or the sugar mayprovide an increase in efficiency. Phophorothioate is used to modify thephosphodiester linkage. An N3′-P5′ phosphoramidate linkage has beendescribed as stabilizing oligonucleotides to nucleases and increasingthe binding to RNA. Peptide nucleic acid (PNA) linkage is a completereplacement of the ribose and phosphodiester backbone and is stable tonucleases, increases the binding affinity to RNA, and does not allowcleavage by RNAse H. Its basic structure is also amenable tomodifications that may allow its optimization as an antisense component.With respect to modifications of the heterocycle, certain heterocyclemodifications have proven to augment antisense effects withoutinterfering with RNAse H activity. An example of such modification isC-5 thiazole modification. Finally, modification of the sugar may alsobe considered. 2′-O-propyl and 2′-methoxyethoxy ribose modificationsstabilize oligonucleotides to nucleases in cell culture and in vivo.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is anacronym for DNA loci that contain multiple, short, direct repetitions ofbase sequences.

The prokaryotic CRISPR/Cas system has been adapted for use as geneediting (silencing, enhancing or changing specific genes) for use ineukaryotes (see, for example, Cong, Science, 15:339(6121):819-823 (2013)and Jinek, et al., Science, 337(6096):816-21 (2012)). By transfecting acell with elements including a Cas gene and specifically designedCRISPRs, nucleic acid sequences can be cut and modified at any desiredlocation. Methods of preparing compositions for use in genome editingusing the CRISPR/Cas systems are described in detail in US Pub. No.2016/0340661, US Pub. No. 20160340662, US Pub. No. 2016/0354487, US Pub.No. 2016/0355796, US Pub. No. 20160355797, and WO 2014/018423, which arespecifically incorporated by reference herein in their entireties.

Thus, as used herein, “CRISPR system” refers collectively to transcriptsand other elements involved in the expression of or directing theactivity of CRISPR-associated (“Cas”) genes, including sequencesencoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g.,tracrRNA or an active partial tracrRNA), a tracr-mate sequence(encompassing a “direct repeat” and a tracrRNA-processed partial directrepeat in the context of an endogenous CRISPR system), a guide sequence(also referred to as a “spacer”, “guide RNA” or “gRNA” in the context ofan endogenous CRISPR system), or other sequences and transcripts from aCRISPR locus. One or more tracr mate sequences operably linked to aguide sequence (e.g., direct repeat-spacer-direct repeat) can also bereferred to as “pre-crRNA” (pre-CRISPR RNA) before processing or crRNAafter processing by a nuclease.

There are many resources available for helping practitioners determinesuitable target sites once a desired DNA target sequence is identified.For example, numerous public resources, including a bioinformaticallygenerated list of about 190,000 potential sgRNAs, targeting more than40% of human exons, are available to aid practitioners in selectingtarget sites and designing the associate sgRNA to affect a nick ordouble strand break at the site. See also, crispr.u-psud.fr, a tooldesigned to help scientists find CRISPR targeting sites in a wide rangeof species and generate the appropriate crRNA sequences.

Inhibitory nucleic acids, such as siRNA, shRNA, ribozymes, or antisensemolecules, can be synthesized and introduced into cells using methodsknown in the art. Molecules can be synthesized chemically orenzymatically in vitro (Micura, Agnes Chem. Int. Ed. Emgl. 41 2265-9(2002); Paddison et al., Proc. Natl. Acad. Sci. USA, 99:1443-8 2002) orendogenously expressed inside the cells in the form of shRNAs (Yu etal., Proc. Natl. Acad. Sci. USA, 99:6047-52 (2002); McManus et al., RNA8, 842-50 (2002)). Plasmid-based expression systems using RNA polymeraseIII U6 or H1, or RNA polymerase II U1, small nuclear RNA promoters, havebeen used for endogenous expression of shRNAs (Brummelkamp et al.,Science, 296: 550-3 (2002); Sui et al., Proc. Natl. Acad. Sci. USA, 99:5515-20 (2002); Novarino et al., J. Neurosci., 24: 5322-30 (2004)).Synthetic siRNAs can be delivered by electroporation or by usinglipophilic agents (McManus et al., RNA 8, 842-50 (2002); Kishida et al.,J. Gene Med., 6: 105-10 (2004)). Alternatively, plasmid systems can beused to stably express small hairpin RNAs (shRNA) for the suppression oftarget genes (Dykxhoorn et al., Nat. Rev. Mol. Biol., 4:457-67 (2003)).Various viral delivery systems have been developed to delivershRNA-expressing cassettes into cells that are difficult to transfect(Brummelkamp et al., Cancer Cell, 2: 243-7 (2002); Rubinson et al., Nat.Genet., 33: 401-6 2003). Furthermore, siRNAs can also be delivered intolive animals. (Hasuwa et al., FEBS Lett., 532, 227-30 (2002); Carmell etal., Nat. Struct. Biol., 10: 91-2 (2003); Kobayashi et al., J.Pharmacol. Exp. Ther., 308:688-93 (2004)).

Inhibitory oligonucleotides can be delivered to a cell by directtransfection or transfection and expression via an expression vector.Appropriate expression vectors include mammalian expression vectors andviral vectors, into which has been cloned an inhibitory oligonucleotidewith the appropriate regulatory sequences including a promoter to resultin expression of the antisense RNA in a host cell. Suitable promoterscan be constitutive or development-specific promoters. Transfectiondelivery can be achieved by liposomal transfection reagents, known inthe art (e.g., Xtreme transfection reagent, Roche, Alameda, Calif.;Lipofectamine formulations, Invitrogen, Carlsbad, Calif.). Deliverymediated by cationic liposomes, by retroviral vectors and directdelivery are efficient. Another possible delivery mode is targetingusing antibody to cell surface markers for the target cells.

In some embodiments, one or more vectors driving expression of one ormore elements of a CRISPR system are introduced into a target cell suchthat expression of the elements of the CRISPR system direct formation ofa CRISPR complex at one or more target sites. Accordingly, cleavage ofDNA by the genome editing vector or composition can be used to deletenucleic acid material from a target DNA sequence by cleaving the targetDNA sequence and allowing the cell to repair the sequence. As such, thecompositions can be used to modify DNA in a site-specific, i.e.,“targeted” way, for example gene knock-out, gene knock-in, gene editing,gene tagging, etc., as used in, for example, gene therapy.

While the specifics can be varied in different engineered CRISPRsystems, the overall methodology is similar. A practitioner interestedin using CRISPR technology to target a DNA sequence can insert a shortDNA fragment containing the target sequence into a guide RNA expressionplasmid. The sgRNA expression plasmid contains the target sequence(about 20 nucleotides), a form of the tracrRNA sequence (the scaffold)as well as a suitable promoter and necessary elements for properprocessing in eukaryotic cells. Such vectors are commercially available(see, for example, Addgene). Many of the systems rely on custom,complementary oligos that are annealed to form a double stranded DNA andthen cloned into the sgRNA expression plasmid. Co-expression of thesgRNA and the appropriate Cas enzyme from the same or separate plasmidsin transfected cells results in a single or double strand break(depending of the activity of the Cas enzyme) at the desired targetsite.

In another aspect, the present invention provides a method ofameliorating NLRP3 inflammasome-associated inflammatory and/ordegenerative diseases in a subject. As used herein, the term“ameliorate” means that the clinical signs and/or the symptomsassociated with NLRP3 inflammasome-associated inflammatory and/ordegenerative diseases are lessened. The signs or symptoms to bemonitored will be characteristic of a particular disease or disorder andwill be well known to the skilled clinician, as will the methods formonitoring the signs and conditions thereof.

In another aspect, the present invention provides a method ofidentifying an agent useful for treating a NLRP3 inflammasome-associatedinflammatory and/or degenerative disease or disorder through thetargeting of IRF1 and/or CMPK2. The method includes contacting a sampleof cells with at least one test agent, wherein a decrease in NLPR3inflammasome activation in the presence of the test agent as compared toNLPR3 inflammasome activation in the absence of the test agentidentifies the agent as useful for treating NLRP3inflammasome-associated inflammatory and degenerative diseases. In oneembodiment, a decrease in IRF1 activity or expression and/or a decreasein CMPK2 activity or expression in the presence of the test agent ascompared to IRF1 activity or expression and/or CMPK2 activity orexpression in the absence of the test agent identifies the agent asuseful for treating a NLRP3 inflammasome-associated inflammatory anddegenerative disease. In various embodiments, the method may beperformed in a high throughput format, such as contacting samples ofcells of a plurality of samples with at least one test agent. In variousembodiments, the plurality of samples may be obtained from a singlesubject or from different subjects.

An agent useful in a method of the invention can be any type ofmolecule, for example, a polynucleotide, a peptide, antisenseoligonucleotide, antibody or antibody fragment, a peptidomimetic,peptoids such as vinylogous peptoids, a small organic molecule, anucleotide analog, or the like, and can act in any of various ways toreduce or inhibit elevated NLPR3 inflammasome activation, IRF1 activityor expression, and/or CMPK2 activity or expression. In variousembodiments, the inhibitor of inhibitor of CMPK2 activity or expressionis an inhibitory nucleic acid that inhibits the expression of CMPK2. Forexample, the inhibitory nucleic acid can be siRNA, shRNA, guide RNA(gRNA), oligonucleotides, antisense RNA or ribozymes that inhibit CMPK2synthesis.

Further, the agent can be administered in any way typical of an agentused to treat the particular type of above-mentioned diseases or underconditions that facilitate contact of the agent with the target diseasedcells and, if appropriate, entry into the cells. Entry of apolynucleotide agent into a cell, for example, can be facilitated byincorporating the polynucleotide into a viral vector that can infect thecells. Thus, the inhibitory nucleic acid can be delivered in, forexample, a lentiviral vector, a herpesvirus vector or an adenoviralvector.

If a viral vector specific for the cell type is not available, thevector can be modified to express a receptor (or ligand) specific for aligand (or receptor) expressed on the target cell, or can beencapsulated within a liposome, which also can be modified to includesuch a ligand (or receptor). A peptide agent can be introduced into acell by various methods, including, for example, by engineering thepeptide to contain a protein transduction domain such as the humanimmunodeficiency virus TAT protein transduction domain, which canfacilitate translocation of the peptide into the cell.

Generally, the agent is formulated in a composition (e.g., apharmaceutical composition) suitable for administration to the subject,which can be any vertebrate subject, including a mammalian subject(e.g., a human subject). Such formulated agents are useful asmedicaments for treating a subject suffering from any of theabove-mentioned diseases, in part, by elevated or abnormally elevatedNLRP3 inflammasome activation.

Pharmaceutically acceptable carriers useful for formulating an agent foradministration to a subject are well known in the art and include, forexample, aqueous solutions such as water or physiologically bufferedsaline or other solvents or vehicles such as glycols, glycerol, oilssuch as olive oil or injectable organic esters. A pharmaceuticallyacceptable carrier can contain physiologically acceptable compounds thatact, for example, to stabilize or to increase the absorption of theconjugate. Such physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the physico-chemical characteristics of thetherapeutic agent and on the route of administration of the composition,which can be, for example, orally or parenterally such as intravenously,and by injection, intubation, or other such method known in the art. Thepharmaceutical composition also can contain a second (or more)compound(s) such as a diagnostic reagent, nutritional substance, toxin,or therapeutic agent, for example, a cancer chemotherapeutic agentand/or vitamin(s).

In general, a suitable daily dose of a compound/inhibitor of theinvention will be that amount of the compound/inhibitor that is thelowest dose effective to produce a therapeutic effect. Such an effectivedose will generally depend upon the factors described above. Generally,intravenous, intracerebroventricular and subcutaneous doses of thecompounds of this invention for a patient will range from about 0.0001to about 100 mg per kilogram of body weight per day which can beadministered in single or multiple doses.

When practiced as an in vitro assay, the methods can be adapted to ahigh throughput format, thus allowing the examination of a plurality(i.e., 2, 3, 4, or more) of cell samples and/or test agents, whichindependently can be the same or different, in parallel. A highthroughput format provides numerous advantages, including that testagents can be tested on several samples of cells from a single patient,thus allowing, for example, for the identification of a particularlyeffective concentration of an agent to be administered to the subject,or for the identification of a particularly effective agent to beadministered to the subject. As such, a high throughput format allowsfor the examination of two, three, four, etc., different test agents,alone or in combination, on the macrophages of a subject such that thebest (most effective) agent or combination of agents can be used for atherapeutic procedure. Further, a high throughput format allows, forexample, control samples (positive controls and or negative controls) tobe run in parallel with test samples, including, for example, samples ofcells known to be effectively treated with an agent being tested.

A high throughput method of the invention can be practiced in any of avariety of ways. For example, different samples of cells obtained fromdifferent subjects can be examined, in parallel, with same or differentamounts of one or a plurality of test agent(s); or two or more samplesof cells obtained from one subject can be examined with same ordifferent amounts of one or a plurality of test agent. In addition, cellsamples, which can be of the same or different subjects, can be examinedusing combinations of test agents and/or known effective agents.Variations of these exemplified formats also can be used to identify anagent or combination of agents useful for treating any of theabove-mentioned diseases associated with NLRP3 inflammasome activation.

When performed in a high throughput (or ultra-high throughput) format,the method can be performed on a solid support (e.g., a microtiterplate, a silicon wafer, or a glass slide), wherein samples to becontacted with a test agent are positioned such that each is delineatedfrom each other (e.g., in wells). Any number of samples (e.g., 96, 1024,10,000, 100,000, or more) can be examined in parallel using such amethod, depending on the particular support used. Where samples arepositioned in an array (i.e., a defined pattern), each sample in thearray can be defined by its position (e.g., using an x-y axis), thusproviding an “address” for each sample. An advantage of using anaddressable array format is that the method can be automated, in wholeor in part, such that cell samples, reagents, test agents, and the like,can be dispensed to (or removed from) specified positions at desiredtimes, and samples (or aliquots) can be monitored, for example, forNLRP3 inflammasome activation and/or cell viability.

The invention also provides a method of determining whether any of theabove-mentioned NLRP3 inflammasome-associated inflammatory anddegenerative disease or disorder is amenable to treatment with aninhibitor of NLRP3 inflammasome activation, as disclosed herein. Themethod can be performed, for example, by measuring the amount of NLRP3inflammasome activation, the level of expression or activity of IRF1,and/or the level of expression or activity of CMPK2 in a cell sample ofa subject to be treated, and determining that the measured NLRP3inflammasome activation, expression or activity of IRF1, and/orexpression or activity of CMPK2 is elevated or abnormally elevated ascompared to the level of NLRP3 inflammasome activation, expression oractivity of IRF1, and/or expression or activity of CMPK2 incorresponding normal cells, which can be a sample of normal (i.e., notdiseased) cells of the subject having any one of the above-mentionedinflammatory and/or degenerative diseases. Detection of elevated orabnormally elevated level of NLRP3 inflammasome activation expression oractivity of IRF1, and/or expression or activity of CMPK2 in the cells ascompared to the corresponding normal cells indicates that the subjectcan benefit from treatment with an inhibitor of NLRP3 inflammasomeactivation. A sample of cells used in the present method can be obtainedusing a biopsy procedure (e.g., a needle biopsy), or can be a sample ofcells obtained by a surgical procedure to remove and/or debulk thetumor.

In various embodiments, the method of identifying a disease or disorderamenable to treatment with an inhibitor of NLRP3 inflammasome activationcan further include contacting cells of the sample with at least onetest agent known to inhibit NLRP3 inflammasome activation, IRF1expression or activity, and/or CMPK2 expression or activity, anddetecting a decrease in NLRP3 inflammasome activation or IL-1β releasein the cells following said contact. Such a method provides a means toconfirm that any of the above-mentioned diseases or disorders isamenable to treatment with an inhibitor of NLRP3 inflammasomeactivation. Further, the method can include testing one or moredifferent test agents, either alone or in combination, thus providing ameans to identify one or more test agents useful for treating theparticular symptoms of any of the above-mentioned diseases or disordersbeing examined. Accordingly, the present invention provides a method ofidentifying an agent useful for treating lupus, gout, osteoarthritis,rheumatoid arthritis, ankylosing spondylitis, uveitis, Alzheimer'sdisease, Parkinson's disease, cryopyrin-associated periodic syndromes,type 2 diabetes, atherosclerosis, macular degeneration, lung cancer, andmany more inflammatory and degenerative diseases in a subject.

Amino acid sequences and nucleic acid sequences for human cytidinemonophosphate kinase 2 (CMPK2) and human interferon regulatory factor(IFR1) are known in the art. See, for example, Accession No.: Q5EMB0,human CMPK2, Isoform 1, which provides the amino acid sequence (SEQ IDNO: 1):

MAFARRLLRGPLSGPLLGRRGVCAGAMAPPRRFVLELPDCTLAHFALGADAPGDADAPDPRLAALLGPPERSYSLCVPVTPDAGCGARVRAARLHQRLLHQLRRGPFQRCQLLRLLCYCPGGQAGGAQQGFLLRDPLDDPDTRQALLELLGACQEAPRPHLGEFEADPRGQLWQRLWEVQDGRRLQVGCAQVVPVPEPPLHPVVPDLPSSVVFPDREAARAVLEECTSFIPEARAVLDLVDQCPKQIQKGKFQVVAIEGLDATGKTTVTQSVADSLKAVLLKSPPSCIGQWRKIFDDEPTIIRRAFYSLGNYIVASEIAKESAKSPVIVDRYWHSTATYAIATEVSGGLQHLPPAHHPVYQWPEDLLKPDLILLLTVSPEERLQRLQGRGMEKTREEAELEANSVFRQKVEMSYQRMENPGCHVVDASPSREKVLQTVLSLIQNSFSEPAccession No.: P10914, human interferon regulatory factor (IFR1), whichprovides the amino acid sequence (SEQ ID NO: 2):

MPITRMRMRPWLEMQINSNQIPGLIWINKEEMIFQIPWKHAAKHGWDINKDACLFRSWAIHTGRYKAGEKEPDPKTWKANFRCAMNSLPDIEEVKDQSRNKGSSAVRVYRMLPPLTKNQRKERKSKSSRDAKSKAKRKSCGDSSPDTFSDGLSSSTLPDDHSSYTVPGYMQDLEVEQALTPALSPCAVSSTLPDWHIPVEVVPDSTSDLYNFQVSP1VIPSTSEATTDEDEEGKLPEDIMKLLEQSEWQPTNVDGKGYLLNEPGVQPTSVYGDFSCKEEPEIDSPGGDIGLSLQRVFTDLKNMDATWLDSLLTPVRLPSIQAIPCAP

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE 1 LPS Induces TRIF-Dependent mtDNA Replication in Macrophages

To demonstrate a role for mitochondria in NLRP3 inflammasome activation,it was decided to use low dose ethidium bromide (EtBr) to deplete cellsof mtDNA (Nakahira et al., 2011; Zhong et al., 2016; Shimada et al.,2010). To firmly establish the role of mtDNA in NLRP3 inflammasomeactivation using a genetic approach, it was decided to cross Tfam^(F/F)mice¹⁶ with LysM-Cre mice to generate Tfam^(ΔMye) mice, whichspecifically lack TFAM (transcription factor A, mitochondrial), aprotein that binds mtDNA to promote its compaction and stabilization aswell as replication and transcription (Kang et al. 2007), in maturemyeloid cells. Tfam ablation dramatically reduced mtDNA copy number inbone marrow-derived macrophages (BMDM) (FIG. 6A and FIG. 6B).TFAM-deficient macrophages also failed to produce Ox-mtDNA in responseto treatment with NLRP3 inflammasome activators and displayed defectivecaspase-1 activation and IL-1β processing, whilst retaining expressionof pro-IL-1β and NLRP3 inflammasome components, normal AIM2 inflammasomeactivation in response to poly(dA:dT) and unaltered TNF expression(FIGS. 6C-6G). To rule out the possibility that TFAM itself rather thanmtDNA is required for NLRP3 inflammasome activation, a D-loop (origin ofreplication) containing fragment of mouse mtDNA was amplified in thepresence of the oxidized nucleotide 8-OH-dGTP to generate Ox-mtDNA.Transfection of this Ox-mtDNA into Tfam^(ΔMye) BMDM restored LPS-inducedIL-1β production (FIG. 1A). The effect was NLRP3 dependent, thusdemonstrating that TFAM promotes NLRP3 inflammasome activation byfacilitating Ox-mtDNA formation or release. Last but not least, it wasconfirmed that Ox-mtDNA indeed associated with inflammasome aggregatesupon treatment with NLRP2 inflammasome activators (FIG. 6H).

Since TFAM is required for mtDNA replication and maintenance, whetherLPS-induced “priming” or stimulation with NLRP3 inflammasome activatorsaffected mtDNA abundance was examined. Surprisingly, macrophagestimulation with LPS resulted in a rather rapid increase in mtDNA copynumber (FIG. 1B), correlating with induction of EdU(5-ethynyl-2′-deoxyuridine) incorporation into cytoplasmic organelles(FIG. 1C). LPS-enhanced mtDNA replication was prevented by ablation ofthe Polg gene, which encodes DNA polymerase y (Hudson, G. and Chinnery,P. F., 2006), the enzyme responsible for mtDNA replication (FIG. 1D).Since LPS binds TLR4, which signals via the adaptor proteins MyD88 andTRIF (Kawai, T. and Akira, S. 2010), it was decided to test theirinvolvement in LPS-induced mtDNA replication. Ablation of TRIF preventedLPS-induced mtDNA replication (FIG. 1E). Ablation of MyD88 had a smallereffect, mainly at earlier time points. Ablation of both TRIF and MyD88resulted in complete inhibition of LPS-stimulated mtDNA synthesis.

EXAMPLE 2 IRF1 Controls mtDNA Replication and NLPR3 InflammasomeActivation

To identify how TLR signaling controls LPS-induced mtDNA replication, itwas decided to examine members of the interferon regulatory factor (IRF)family and found that a deficiency in IRF1 obliterated LPS-induced mtDNAreplication (FIGS. 2A-2D). Although resting macrophages barely expressedIRF1, LPS stimulation resulted in robust IRF1 mRNA and protein induction(FIGS. 7A-7C). IRF1 was also needed for NLRP3 inflammasome activation,with Irf1^(−/−) BMDM showing a substantial (50˜70%) reduction in “NLRP3agonist”-induced caspase-1 activation and IL-1β release, without aneffect on AIM2 inflammasome activation (FIG. 2E and 2F). Expression ofNLRP3 inflammasome components and pro-IL-1β and TNF secretion were alsounaffected by IRF1 ablation, which also had no effect on NLRP3agonist-induced mitochondrial damage or mtROS production (FIG. 7D-7G),but due to defective LPS-induced mtDNA replication, less Ox-mtDNA wasfound in Irf1^(−/−) BMDM after “NLRP3 agonist” stimulation (FIG. 2G).These observations suggest that IRF1 controls Ox-mtDNA production andNLRP3 inflammasome activation through its effect on mtDNA replication.

EXAMPLE 3 CMPK2 Controls mtDNA Replication and NLRP3 InflammasomeActivation

Since IRF1 is a transcription factor, it was decided to investigatewhether any of its previously described target genes may be involved inmtDNA replication and found that the IRF1 transcriptome (Jehl et al.,2010) included the gene coding for UMP-CMPK2 (hereafter referred to as“CMPK2”), a mitochondrial deoxyribonucleotide kinase (Ullah et al.,2012). Of note, LPS priming resulted in strong induction of CMPK2 mRNAand protein, whose expression was barely detectable in restingmacrophages (FIG. 3A and FIG. 3B). Importantly, LPS-induced CMPK2upregulation was IRF1 dependent, and Trif^(−/−) and Ifnar1^(−/−) BMDMalso failed to upregulate CMPK2 upon LPS priming (FIGS. 8A-8C). TheCmpk2 promoter contained IRF1 binding sites. CMPK2 is amitochondria-resident nucleotide kinase that is part of the salvagepathway for mitochondrial dNTP synthesis (Xu et al., 2008).Intriguingly, other nucleoside/nucleotide kinases in this pathway andPolγ were not LPS-inducible (FIG. 9), suggesting that CMPK2 is therate-limiting enzyme that controls the supply of dNTP precursors forLPS-induced mtDNA synthesis. CMPK2 phosphorylates dCMP to generate dCDP,which is further converted into dCTP by the mitochondrialdeoxyribonucleotide kinase NME4 (Xu et al., 2008; Milon et al., 2000).Unlike CMPK2, NME4 was expressed constitutively and was not LPSresponsive.

To test the role of CMPK2 in mtDNA replication and NLRP3 inflammasomeactivation, we knocked down Cmpk2 using shRNA. CMPK2-deficientmacrophages (shCmpk2 BMDM) exhibited minimal NLRP3 inflammasomeactivator-induced caspase-1 activation and IL-1β maturation incomparison with CMPK2-sufficient cells (shCtrl), while retaining normalAIM2 inflammasome activation (FIG. 3C and FIG. 3D). Expression of NLRP3inflammasome components and pro-IL-1β was unaffected (FIG. 10A).Although CMPK2 ablation did not affect the extent of mitochondrialdamage or mtROS production after exposure to NLRP3 inflammasomeactivators (FIG. 10B and FIG. 10C), shCmpk2 BMDM failed to upregulatemtDNA replication after LPS stimulation and barely produced Ox-mtDNA(FIGS. 3E and FIG. 3F).

To test whether inactivation of other components of the mitochondrialnucleotide salvage pathway also inhibits NLRP3 inflammasome activation,we knocked down Nme4. shNme4 BMDM failed to replicate mtDNA after LPSpriming, exhibited much lower Ox-mtDNA production than WT BMDM andconsequently secreted much less IL-1β after stimulation with NLRP3inflammasome activators (FIGS. 11A-11C). NME4 silencing, however, didnot affect AIM2 inflammasome activation nor LPS-induced TNF productionand did not diminish CMPK2 induction (FIGS. 11C and 11D).

EXAMPLE 4 CMPK2 Catalytic Activity is Required for IRF1-Dependent NLRP3Inflammasome Activation

To confirm that CMPK2 expression promotes NLRP3 inflammasome activationby providing dCTP for mtDNA synthesis, it was decided to generate acatalytically inactive CMPK2 variant, CMPK2(D330A), by replacing thehighly conserved aspartate (D) residue in its catalytic pocket (Xu etal., 2008; Chen et al., 2008) with alanine (A). Expression of WT CMPK2Irf1^(−/−) BMDM restored LPS-stimulated mtDNA replication, but did notenhance it beyond its normal level when expressed in WT BMDM (FIG. 4A).Although CMPK2 reconstitution did not alter expression of pro-IL-1β,NLRP3, ASC, and pro-caspase-1 and had no effect on induction ofmitochondrial damage by NLRP3 inflammasome activators (FIG. 12B-12C), itrestored Ox-mtDNA production and NLRP3 inflammasome activation (FIG.4A-4B). By contrast, re-expression of CMPK2(D330A) did not restoreLPS-induced mtDNA synthesis, Ox-mtDNA production or NLRP3 inflammasomeactivation, although it did not block these responses when expressed inWT BMDM (FIG. 4C-4D). The data presented herein show strong support thenotion that induction of new mtDNA replication, which depends on CMPK2catalytic activity, is required for production of Ox-mtDNA bymitochondria that have been damaged upon macrophage exposure to “NLRP3agonists”, with Ox-mtDNA being responsible for subsequent NLRP3inflammasome activation.

EXAMPLE 5 Newly Synthesized mtDNA Associates with NLRP3 UponMitochondrial Damage

To further determine the role of newly synthesized mtDNA in NLRP3inflammasome activation, it was decided to incubate WT BMDM with BrdU(bromo-deoxyuridine) to label newly synthesized mtDNA. It was decided tostimulate the cells with LPS+ATP or LPS+nigericin and immunoprecipitatedinflammasomes with antibodies to ASC. The resulting immunecomplexes werepositive for both NLRP3 and BrdU labeled DNA (FIG. 5). However, when ASCwas immunoprecipitated from BMDM that were stimulated with the AIM2agonist poly(dA:dT) neither NLRP3 nor BrdU were present in theimmunecomplexes. To visualize the interaction between newly synthesizedmtDNA and the NLRP3 inflammasome, the cellular localization ofASC-containing inflammasome aggregates and EdU-labeled mtDNA wereexamined before and after NLRP3 agonist treatment. Remarkably, the NLRP3activators ATP and nigericin induced co-localization of newlysynthesized mtDNA and ASC specks, whereas the AIM2 agonist poly(dA:dT)failed to do so. The data presented herein show that the resultstogether with the above genetic and biochemical data collectivelysuggest that newly synthesized mtDNA is more easily cleaved and oxidizedand once released from damaged mitochondria it binds to NLRP3 andpromotes inflammasome activation.

EXAMPLE 6 IRF1 is Required for mtDNA Replication and NLRP3 InflammasomeActivation In Vivo

It was decided to examine the requirement of IRF1 signaling for NLRP3inflammasome activation in vivo. Intraperitoneal (i.p.) injection of LPSis sufficient for induction of an IL-1β and NLRP3-dependent acutesystemic inflammation that eventually leads to death (Lamkanfi andDixit, 2012; Martinon et al., 2009). Relative to WT mice, Irf1^(−/−)mice exhibited drastically reduced IL-1β secretion but little change inTNF production and were largely resistant to LPS-induced death (FIGS.13A-13C). Importantly, Irf1^(−/−) peritoneal macrophages isolated 3 hrsafter LPS injection exhibited lower mtDNA copy number than macrophagesfrom WT mice (FIG. 13D). Irf1^(−/−) mice were also defective inalum-induced IL-1β production and consequently exhibited reducedneutrophil and monocyte infiltration relative to WT counterparts (FIG.13E). Similarly, Irf1^(−/−) mice were also resistant to folicacid-induced acute kidney injury (FIG. 13F), whose pathologicaldevelopment is NLRP3 inflammasome-dependent (Subramanian et al., 2013).

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Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A method of identifying an agent that inhibitsNLRP3 inflammasome activation, comprising contacting a sample of cellswith at least one test agent, wherein a decrease in CMPK2 activity orexpression in the presence of the test agent as compared to CMPK2activity or expression in the absence of the test agent identifies theagent as useful for inhibiting NLRP3 inflammasome activation.
 2. Themethod of claim 1, wherein the test agent is a small molecule,nucleotide analog, peptide, antisense oligonucleotide, antibody orantibody fragment.
 3. The method of claim 1, wherein a decrease in IRF1or CMPK2 activity or expression in the presence of the test agent ascompared to IRF 1 or CMPK2 activity or expression in the absence of thetest agent identifies the agent as useful for treating NLRP3inflammasome-associated inflammatory and degenerative diseases.
 4. Themethod of claim 3, wherein the NLRP3 inflammasome-associatedinflammatory and/or degenerative disease is selected from the groupconsisting of cancer, lupus, gout, rheumatoid arthritis, osteoarthritis,ankylosing spondylitis, uveitis, Alzheimer's disease, Parkinson'sdisease, cryopyrin-associated periodic syndromes, nonalcoholicsteatohepatitis (NASH), type 2 diabetes, atherosclerosis, and maculardegeneration.
 5. The method of claim 1, which is performed in a highthroughput format.
 6. The method of claim 5, comprising contacting aplurality of samples of cells with at least one test agent.
 7. Themethod of claim 6, wherein the plurality of samples is obtained from asingle subject.
 8. The method of claim 6, wherein the plurality ofsamples is obtained from different subjects.
 9. The method of claim 6,comprising contacting a sample of cells with a plurality of test agents.